Assay for porcine circovirus production

ABSTRACT

The present invention provides methods for the determination of the viral titer of a culture of host animal host cells infected with a circovirus. The FACS-based methods of the invention may include determining the viability of the host cells in a cell culture medium supernatant and of those cells that remain adherent to a solid support. Detecting and measuring the percentage of cells that expressed the viral antigens ORF1 and ORF2 may determine the viral load of the cultured host cells. The yield of the virus may be established by the detection and measurement of both antigens in supernatant cells, for example 5 to 7 days from when the host cells are transferred to a serum free medium. The methods of the invention may yield rapid quantitative data. This allows the repeated in-process monitoring of the viral production throughout the incubation period, and ready selection of the most appropriate harvesting point.

RELATED APPLICATIONS

This application claims priority to Provisional U.S. Application Ser.No. 60/670,892 filed Apr. 13, 2005, the contents of which are herebyexpressly incorporated herein by reference.

INCORPORATION BY REFERENCE

All documents cited therein or during their prosecution (“applicationcited documents”) and all documents cited or referenced in theapplication cited documents, and all documents cited or referencedherein (“herein cited documents”), and all documents cited or referencedin herein cited documents, together with any manufacturer'sinstructions, descriptions, product specifications, and product sheetsfor any products mentioned herein or in any document incorporated byreference herein, are hereby incorporated herein by reference, and maybe employed in the practice of the invention

FIELD OF THE INVENTION

This invention relates to methods of in-process monitoring of a viralyield during batch incubation production processes using fluorescentantibody cell sorting analysis. This invention further relates tomethods of in-process monitoring and harvesting of circovirus infectedcell cultures in medium that may lack a serum component.

BACKGROUND OF THE INVENTION

Postweaning multisystemic wasting syndrome (PMWS) is a recentlyrecognized disease of young pigs. The PMWS syndrome detected in Canada,the United States and France is clinically characterized by a gradualloss of weight and by manifestations such as tachypnea, dyspnea andjaundice. From the pathological point of view, it is manifested bylymphocytic or granulomatous infiltrations, lymphadenopathies and, morerarely, by hepatitis and lymphocytic or granulomatous nephritis (ClarkE. G. (1997) Proc. Am. Assoc. Swine Prac. 499-501; La SemaineVeterinaire No. 26, supplement to La Semaine Veterinaire 1996 (834); LaSemaine Veterinaire 1997 (857): 54; Nayar et al. (1997) Can. Vet. J.38:385-387). Treatment and prevention of this disease are not currentlyavailable. Several lines of evidence, however, point to porcinecircovirus as the etiologic agent of PMWS (Ellis et al. (1998) Can. Vet.J. 39:44-51). Circoviruses have been recovered from pigs with PMWS, andantibodies to porcine circovirus have been demonstrated in pigs with thedisease.

A family of viruses, named Circoviridae, found in a range of plant andanimal species and commonly referred to as circoviruses, arecharacterized as round, non-enveloped virions with mean diameters from17 to 23.5 nm containing circular, single-stranded deoxyribonucleic acid(ssDNA). The ssDNA genome of the circoviruses represent the smallestviral DNA replicons known. As disclosed in WO 99/45956, at least sixviruses have been identified as members of the family according to TheSixth Report of the International Committee for the Taxonomy of Viruses(Lukert et al. 1995, The Circoviridae, pp. 166-168. In Murphy, et al.(eds.) Virus Taxonomy, Sixth Report of the International Committee onTaxonomy of Viruses, Arch. Virol. 10 Suppl.).

Animal viruses included in the family are chicken anemia virus (CAV);beak and feather disease virus (BFDV); porcine circovirus (PCV); andpigeon circovirus. PCV was originally isolated from porcine kidney cellcultures. PCV replicates in the cell nucleus and produces largeintranuclear inclusion bodies. See Murphy et al. (1999, Circoviridae p.357-361, Veterinary Virology, 3rd ed. Academic Press, San Diego). Thereare currently two recognized types of PCV, PCV type 1 (PCV1) and PCVtype 2 (PCV2). PCV1, isolated as a persistent contaminant of thecontinuous porcine kidney cell line PK-15 (ATCC CCL31), does not causedetectable cytopathic effects in cell culture and fails to produceclinical disease in pigs after experimental infection (see Allan G.(1995) Vet. Microbiol. 44: 49-64; Tischer et al. (1982) Nature295:64-66; and Tischer et al. (1986) Arch. Virol 91:271-276).

It is only very recently that some authors have thought that strains ofPCV could be pathogenic and associated with the PMWS syndrome (Nayar etal. (1997) Can. Vet. J. 38: 385-387 and Clark E. G. (1997) Proc. Am.Assoc. Swine Prac. 499-501). Nayar et al. have detected PCV DNA in pigshaving the PMWS syndrome using PCR techniques. PCV2, in contrast toPCV1, is closely associated with post weaning multisystemic wastingsyndrome (PMWS) in weanling pigs (see Allan et al. (1998) Eur. J. Vet.Diagn. Investig. 10:3-10; Ellis et al. (1998) Can. Vet. J. 39:44-51 andMorozov et al. (1998), J. Clin. Microbiol. 36:2535-2541).

The nucleotide sequences for PCV1 are disclosed in Mankertz et al.(1997) J. Virol. 71:2562-2566) and Meehan et al. (1997) J. Gen. Virol.78:221-227) and the nucleotide sequences for PCV2 are disclosed in Hamelet al. (1998) J. Virol. 72:5262-5267; Mankertz et al. (2000) Virus Res.66:65-77 and Meehan et al. (1998) J. Gen. Virol. 79:2171-2179. Strainsof PCV2 are disclosed in WO 00/01409 and have been deposited at theEuropean Collection of Cell Cultures, Centre for Applied Microbiology &Research, Porton Down, Salisbury, Wiltshire SP4 OJG, United Kingdom andinclude: Accession No. V97100219; Accession No. V9700218; Accession No.V97100217; Accession No. V98011608; and Accession No. V98011609. WO00/77216 also discloses PCV2.

As many as thirteen open reading frames (ORFs) have been identified inthe PCV2 genome. ORF1 (Meehan et al., (1998)), alternatively designatedas ORF4, comprises the nucleotides 398-1342 (GenBank Accession No.AF055392) and has the potential to encode a protein with a predictedmolecular weight of 37.7 kD. ORF2 (Meehan et al., (1998); alternativelydesignated as ORF13, comprises the nucleotides 1381-1768 joined to 1-314(GenBank Accession No. AF055392) and may encode a protein with apredicted molecular weight of 27.8 kD. Further description of the PCV2ORFs 1-13 may be found in U.S. Pat. Nos. 6,368,601, 6,391,314, 6,660272,6,217,883, 6,517,843, 6,497,883 as well as AU 764379, EP 1019510, MX221562, MX 216996, RU 2237492 and NZ 505008 which are incorporatedherein by reference in the entirety.

ORF1 of PCV2 is highly similar (86% identity) to the ORF1 of the PCV1isolate (Meehan et al., (1998)). The ORF1 protein of PCV1 has beenpartially characterized (Meehan et al., 1997; Mankertz et al., (1998)Virus genes 16:267-276) and has been shown to be essential for virusreplication, most probably involved in the viral DNA replication.

Protein sequence identity between the respective ORF2s was lower (66%identity) than that between the ORF1s, but each of the ORF2s shared ahighly conserved basic N-terminal region, similar to that observed inthe N-terminal region of the major structural protein of the aviancircovirus chicken anemia virus (CAV) (Meehan et al., 1998). Recently,Mankertz et al., in (1998) J. Gen. Virol. 79:381-384 has suggested thatthe ORF2 of the PCV1 isolate (designated ORF1 in Mankertz et al., 1998)codes for a capsid protein. The transcription analysis of the genome ofPCV2 has not been published yet. Recent data obtained with the PCV1isolate indicated that the ORF2 transcript is spliced (Mankertz et al.,1998).

Published studies to date on PCV2 used either tissue homogenate orcultured virus derived from field isolates. Tischer et al. ((1987) ArchVirol. 96:39-57) report that porcine kidney cells are stimulated toentry to the S phase in the cell cycle by D-glucosamine treatment.However, the treatment must be performed with caution becauseD-glucosamine is toxic for cell culture (see, Allan et al. (2000) J.Vet. Diagn. Investigation. 12:3-14).

There is a remaining need for methods for culturing circovirusincluding, for example, PCV1, PCV2 and other circoviruses, such thatcircovirus in high yield is possible. Such methods would beadvantageous, in particular for preparation of PCV2 antigens as vaccinesdirected against PMWS. The present invention addresses that need. Theinvention relates to methods for growing and quantifying the infectiousor antigenic amount and determining antibodies against circoviruses, inparticular porcine circoviruses (PCV) that allow for in-processmonitoring of the progress of the production of the virus in the batchculture.

Although porcine circovirus can be detected as a contaminating agent inpig tissue cultures, successful large-scale batch cultures of the virusrequire rapid assays to allow continual monitoring of the progress ofviral production to obtain optimal yields. The object of the presentinvention, therefore, was to develop a method for monitoring theprogress of the cultivation of a circovirus such as a porcine circovirusin vitro to be able to examine the ORFs kinetic expression. It was alsointended to increase virus yield of a cell culture for the production ofa vaccine that may require inactivated PCV or an avirulent PCV strain(e.g. through selection of an avirulent PCV strain after adaptation tovarious cell cultures and/or after treatment of infected cell cultureswith mutagens or after genetic modification of the PCV) as live vaccine.In addition, the antigenic material obtained from grown porcinecircoviruses can also be employed for diagnostic purposes. There is aneed, therefore, to be able to periodically and rapidly monitor theprogress of a batch cell culture of a circovirus under conditions thatprovide viral particles suitable for vaccine or other purposes. There isa need for monitoring methods that can give rapid results, rather thanthe labor-intensive and time-consuming methods currently employed forthat purpose.

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY OF THE INVENTION

This invention provides a FACS-based procedure for the in-processmonitoring and rapid determination of the useful harvesting point of acell culture infected with a circovirus such that an optimum yield ofthe virus can be obtained. The methods encompass providing a seedculture of circovirus infected host cells and inoculating a batchculture therewith, incubating the seed culture, removing aliquots of thecultured cells, separating supernatant cells and adherent cells,releasing the adherent cells from their substratum, determining theviability of the host cells, and determining the percentage of ORF1- andORF2-positive cells by FACS, thereby determining the harvesting point ofthe culture.

One aspect of the invention provides a FACS-based method for detectingthe production of circovirus antigen by a culture of host cells, whereinthe method may encompass the steps of obtaining from a host cell cultureinfected with a circovirus a sample comprising a population ofnon-adherent host cells and a population of host cells adhering to asubstratum, isolating from the sample the non-attached host cellsthereof, isolating from the sample the adherent host cells and thesubstratum thereof and releasing the adherent host cells from thesubstratum, determining the amount of ORF1 in the non-adherent andreleased adherent host cells by contacting said cells with anORF1-specific antibody, determining the percentage of infected cells inthe non-adherent and released adherent host cells by contacting saidcells with an ORF2-specific antibody, and relating the percentage ofORF1- and ORF2-positive cells in the sample to the amount of circovirusin the sample.

In various embodiments of the method, the circovirus can be PCV2. Invarious embodiments, the host cell strain can be PK-15 or other suitablecell lines. It is contemplated, however, that the methods of theinvention can be usefully applied for detecting and in-processmonitoring of the production of any circovirus cultured on isolated hostcells and for which there are available viral antigen specificantibodies.

Another aspect of the invention provides a FACS-based method forin-process monitoring of the production of a circovirus from culturedhost cells, encompassing the steps of obtaining from a host cell cultureinfected with a circovirus a time-dependent plurality of samples, eachsample in the series comprising a population of non-adherent host cellsand a population of host cells adhering to a substratum, isolating fromeach sample of cell culture the non-attached host cells thereof,isolating from each sample of cell culture the adherent host cells andthe substratum thereof and releasing the adherent host cells from thesubstratum, determining the viability of the host cells in the samplesby measuring propidium iodide uptake using flow cytometry, determiningthe percentage of ORF I-positive cells present in the non-adherent andreleased adherent cells determining the amount of ORF1 in thenon-adherent and released adherent host cells by contacting said cellswith an ORF1-specific antibody, determining the amount of the antibodybinding to the cells by FACS and relating the amount of bound antibodyto the amount of ORF1 present in the cells, determining the percentageof ORF2-positive cells present in the non-adherent and released adherentcells by determining the amount of ORF2 in the non-adherent and releasedadherent host cells by contacting said cells with an ORF2-specificantibody, determining the amount of the antibody binding to the cells byFACS and relating the amount of bound antibody to the amount of ORF2present in the cells, and plotting the changes in the levels ofviability, ORF1 and ORF2, thereby determining the time course of theproduction of the circovirus in the host cell culture.

In one embodiment of this method of the invention, the viability of thecells can be determined with propidium iodide and flow cytometry.

In the various embodiments of this method of the invention, thecircovirus can be PCV2 and the host cell strain can be PK-15, althoughthe methods should not be construed as to be applicable solely to thisstrain of circovirus/host cell combination.

Yet another aspect of the invention is a method of producing circovirusin yields that may be useful, for example, the preparation of a vaccine,encompassing the steps of preparing a seed culture of a circovirus byin-process monitoring of the expression of ORF1 and seeding a host cellculture with a seed culture of a circovirus, incubating the host cellculture in the absence of fetal calf serum, monitoring (i) viability ofthe host cells, and (ii) ORF1 and ORF2 expression by the host cells, andharvesting the circovirus from the host cell culture when ORF2expression is approximately the same in the non-adherent cells andadherent cells of the host cell culture.

In various embodiments of this aspect of the invention, the yield ofcircovirus in the seed culture may be determined by monitoring theexpression of circovirus ORF1 and ORF2 antigens in supernatant hostcells using a fluorescent antibody cell sorting (FACS)-based method, andwherein the seed culture is harvested when ORF2 is expressed by thenon-adherent cells.

In the various embodiments of this aspect of the invention, theviability of the cells may be determined by measuring propidium iodideuptake using flow cytometry.

In the various embodiments of this method of the invention, thecircovirus can be PCV2 and the host cell strain can be PK-15, althoughthe methods should not be construed as to be applicable solely to thisstrain of circovirus/host cell combination. It is contemplated, however,that the methods of the invention can be usefully applied for detectingand in-process monitoring of the production of any circovirus culturedon isolated host cells and for which there are available viral antigenspecific antibodies.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF DRAWINGS

The following detailed description, given by way of examples, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawings, incorporated herein by reference, in which:

FIG. 1 illustrates a schema for the separation of supernatant andadherent PK-15 cells from a cell culture and determination of viralantigens ORF1 and ORF2 therein;

FIG. 2 illustrates the flow cytometry counting of total PK-15 cells in acell suspension, including live and dead cells;

FIG. 3 illustrates the detection of the proportion of dead cells in apopulation of PCV2 infected PK-15 cells by the shift in propidium iodineuptake and FACS;

FIG. 4 illustrates a time course of viability of a working seed cultureof PCV2 infected PK-15 cells with fetal calf serum present throughoutthe incubation period;

FIG. 5 illustrates ORF1 immunodetection and measurement by FACS;

FIG. 6 illustrates the production of the PCV2 viral ORF1 and ORF2antigens during the incubation period of a working seed culture; and

FIG. 7 illustrates the production of the viral antigens ORF 1 and ORF2in PK-15 cells infected with PCV2 virus with removal of FCS from theculture medium after 3 days.

DETAILED DESCRIPTION

Various documents are cited in the following text, and various documentsare referenced or cited in documents cited in the following text. Thereis no admission that any of these documents are indeed prior art as tothe present invention. All documents cited herein and all documentsreferenced or cited in documents cited herein are hereby incorporatedherein by reference.

The term “flow cytometer” as used herein refers to any device that willirradiate a particle suspended in a fluid medium with light at a firstwavelength, and is capable of detecting a light at the same or adifferent wavelength, wherein the detected light indicates the presenceof a cell or an indicator thereon. The “flow cytometer” may be coupledto a cell sorter that is capable of isolating the particle or cell fromother particles or cells not emitting the second light. The indicator onthe cell surface may be an antibody coupled to a fluorophore such as,but not limited to, FITC providing Flourescent Antibody Cell Sorting(FACS).

The term “host cell” as used herein refers to an isolated cell that is ahost for the infection and replication of a virus, preferably acircovirus.

The terms “adherent cells” as used herein refers to animal cells grownin vitro and which have attached to a substratum. The substratum may bethe surface of a culture container or, as in batch cell cultures, may bea particulate solid support such as CYTODEX™ (Amersham Biosciences, Inc)beads or the like. Such cells may be removed from the underlying solidsupport by methods requiring enzymatic digestion of the subcelluarmatrix, including digestion under controlled conditions using a proteasesuch as, but not limited to, trypsin. Conversely, term “non-adherentcells” refers to those cultured cells that have detached from asubstratum during the course of the culture period.

As used herein, genes, gene loci or transcripts thereof have italicizeddesignators; and protein or polypeptides expressed therefrom havenon-italicized designators.

The terms “ORF1” and “ORF2” as used herein refer to circoviral antigensexpressed from the open-reading frames ORF1 and ORF2 (as designated byMeehan et al., (1998) J. Gen. Virol. 78:221-227) respectively. ORF1 isbelieved to be an early-stage replicase and ORF2 a polypeptidecontributing to the viral capsid. Thirteen open reading frames (ORFs)have been identified in the PCV2 genome. Further description of the PCV2ORFs may be found in U.S. Pat. Nos. 6,368,601, 6,391,314, 6,660,272,6,217,883, 6,517,843, 6,497,883 as well as AU 764379, EP 1019510, MX221562, MX 216996, RU 2237492 and NZ 505008 which are incorporatedherein by reference in the entirety. Correspondence between the variousdesignations assigned to each ORF of PCV2 is shown in Example 1 below.As used herein, ORF1 and ORF2 correspond to ORF4 and ORF13 (asdesignated in the above-referenced patents) respectively.

The term “in-process” refers to the monitoring of parameters that arecharacteristic of cell and virus culture, including a virally infectedcell culture, throughout the period of the culture. The monitoring canbe continuous, such as monitoring the pH or oxygen content of theculture medium, or can be periodic monitoring wherein samples arewithdrawn from the culture at selected time points, parameters such asviability or viral antigen content are detected and measured and theparameters are plotted versus the time of the culture.

The term “seed culture” as used herein refers to a culture of host cellsinfected with a selected virus such as a circovirus and which is thenincubated for a period to allow the titer of virus to increase.Typically, but not necessarily, the volume of a seed culture is lessthan the volume of the subsequent main culture or fermentation mediumthat receives the seed culture.

Following longstanding law convention, the terms “a” and “an” as usedherein, including the claims, are understood to mean “one” or “more”.

Abbreviations: ORF, open reading frame; ORF, nucleotide sequenceencoding an ORF; PK, porcine kidney; PCV, Porcine Circo Virus; PK-15,porcine kidney cells; FACS, fluorescent antibody cell sorting; ELISA,enzyme-linked immunosorbant assay; Mab, monoclonal antibody; FITC,Fluorescein isothiocyanate; IgG, immunoglobulin G; PBS/BSA, phosphatebuffered saline/bovine serum albumen.

The present invention provides methods for the determination of theviral yield of a culture of host animal cells infected with acircovirus, in particular a porcine circovirus. The methods of theinvention, however, are generally applicable to any other strain or typeof circovirus growing on cell culture, especially in batch cultureprocedures. The methods of the invention are particularly useful for themonitoring of the viral yield of batch cultures of the porcinecircovirus strain PCV2.

The FACS-based methods of the invention comprise determining theviability of the host cells in a cell culture medium supernatant and ofthose cells that remain adhered to a solid support such as, but notlimited to, CYTODEX™ (Amersham Biosciences, Inc). The viral load of thecultured host cells is measured by determining the percentage ofORF1-positive cells present in the non-adherent and released adherentcells, determining the percentage of ORF2-positive cells present in thenon-adherent and released adherent cells, relating the percentage ofORF1- and ORF2-positive cells in the sample to the amount of circovirusin the sample. The yield of the virus is established by detection andmeasurement of both antigens in supernatant cells, for example 5 to 7days from when the host cells are transferred to a serum free medium.

One embodiment of the invention, therefore, is a novel assay suitablefor the titration of PCV2 based on the immunodetection in PK-15 hostcells of viral protein by flow cytometry using monoclonal antibodiesspecific for either ORF1 or ORF2. Furthermore, the kinetics of PK-15cells growth can also be monitored by flow cytometry. The rapidity ofthe methods of the invention allows for the determination of an optimumharvesting point to achieve high viral yields. A harvesting point can beselected that offers a viral yield useful, for example, for theproduction of a vaccine, while minimizing the incubation period of thecells with attendant cost reductions. The methods of the inventionrapidly yield quantitative data. This allows for the repeated monitoringof the viral production throughout the course of the incubation periodand ready selection of the most appropriate harvesting point.

Traditional methods of measuring viral titer are much slower and involveextended culturing of test samples to form countable plaques.Conventional plate assays of the virus growing on PK-15 cells that arebased on immunofluorescence detection using an ELISA based on an ORF2monoclonal antibody are time consuming and labour intensive, requiringseveral days to obtain a useful result.

One aspect of the invention is a method for rapidly determining theviability characteristics of cultured host cells during the time courseof a batch culture. Although the method is useful and suitable formonitoring the culture of any cells for viral production, the methodsare particularly useful for the in-process control of cell cultures ofPK-15 cells for the production of batches of PCV2 circovirus for vaccineproduction.

In this aspect of the methods according to the invention, and asillustrated in FIG. 1, samples of batch cell cultures are divided into apopulation of supernatant (non-adherent) cells and a population of cellsadherent to a solid substratum. A substratum for use in culturing thehost PK-15 cells, for example, is CYTODEX™ (Amersham Biosciences, Inc)beads, although any other suitable material known to those with skill inthe art of tissue culture can be selected. Dense substratum beads areallowed to settle by gravity and the supernatant culture medium with thenon-adherent cells is removed by any of a variety of methods such asaspiration, decantation, centrifugation and the like. The cells adherentto the substratum may then released from the substratum by, for example,trypsinization, the degree of the digestion release being monitoredunder a microscope to establish the point of maximum release of thecells from the substratum, with minimal damage to the cells themselves.The released cells are collected to yield the second of the two desiredcell populations.

Described herein are methods for the production of the antibodiescapable of specifically recognizing one or more epitopes of the ORF1 orORF2 protein of a circovirus such as, but not limited to, PCV2. Suchantibodies may include, but are not limited to polyclonal antibodies,monoclonal antibodies (mAbs), chimeric antibodies, single chainantibodies, Fab fragments, F(ab′)₂ fragments, fragments produced by aFAb expression library, anti-idiotypic (anti-Id) antibodies, andepitope-binding fragments of any of the above. Advantageously, theantibodies for use in the present invention are monoclonal antibodiesspecific for either the ORF1 or the ORF2 protein expressed by the ORF1and ORF2 genes of a circovirus. Most advantageously, the ORF1 or ORF2proteins are expressed by the circovirus PCV2.

For the production of antibodies, various host animals may be immunizedby injection with an isolated ORF1 or ORF2 polypeptide or an immunogenicpeptide thereof. Such host animals may include but are not limited torabbits, mice, and rats, to name but a few. Various adjuvants may beused to increase the immunologic response, depending on the hostspecies, including but not limited to Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentiallyuseful human adjuvants such as BCG (bacille Calmette-Guerin) andCorynebacterium parvum. Following completion of the immunization steps,antisera reactive with the ORF1 or ORF2 protein may be collected and, ifdesired, polyclonal anti-ORF1 (or anti-ORF2) protein antibodiesisolated.

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen,such as a target gene product, or an antigenic functional derivativethereof. For the production of polyclonal antibodies, host animals suchas those described above, may be immunized by injection with the ORF1 orORF2 protein supplemented with adjuvants as also described above.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, may be obtained by any technique that providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to the hybridoma techniqueof Kohler & Milstein (1975) Nature 256: 495-497; and U.S. Pat. No.4,376,110), the human B-cell hybridoma technique (Kosbor et al. (1983)Immunology Today 4: 72; Cole et al. (1983) Proc. Natl. Acad. Sci. U.S.A.80: 2026-2030), and the EBV-hybridoma technique (Cole et al. (1985)Monoclonal Antibodies And Cancer Therapy Alan R. Liss, Inc. pp. 77-96).Briefly, spleen cells are harvested from an immunized mouse and fusedwith immortalizing cells (i.e., myeloma cells) to yieldantibody-producing hybridomas. Hybridomas can be screenedimmunochemically for production of monoclonal antibodies specificallyreactive with the ORF1 or ORF2 protein. Commercial sources for obtainingcustom polyclonal antisera and monoclonal antibodies are also available.For example, HTI Bio-Products, Inc. (Ramona, Calif.) producescustom-made antibodies, antisera, ascites fluid and hybridoma lines.

Protocols for producing, isolating and purifying conventional andmonoclonal antibodies may be analogous to those described in Cassone etal. (1988) J. Med. Microbiol. 27: 233-238; Hancock & Evan Production andCharacterization of Antibodies against Synthetic Peptides pp 23-33 inImmunochemical Protocols ed. M. M. Manson, (1992) (Humana Press, Totowa,N.J.); Goding, J. W., Monoclonal Antibodies: Principles and Practice, 2ded., (1986) (Academic Press Ltd., London) and Lam & Mutharia,“Antigen-Antibody Reactions,” pp104-132 in Methods for General andMolecular Bacteriology, ed. P. Gerhardt, (1994) (ASM Press, Washington,D.C.) the contents of which are incorporated herein by reference intheir entirety.

Such antibodies may be of any immunoglobulin class including IgG, IgM,IgE, IgA, IgD and any subclass thereof. The hybridoma producing the MAbsof this invention may be cultivated in vitro or in vivo. Production ofhigh titers of MAbs in vivo makes this the presently preferred method ofproduction.

Alternatively, techniques described for the production of single chainantibodies such as, but not only U.S. Pat. No. 4,946,778; Bird (1988)Science 242: 423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. 85:5879-5883; and Ward et al. (1989) Nature 334: 544-546 can be adapted toproduce the ORF1 or ORF2 protein-specific antibodies. Single chainantibodies are formed by linking the heavy and light chain fragments ofthe Fv region via an amino acid bridge, resulting in a single chainpolypeptide.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, such fragments include but are notlimited to: the F(ab′)₂ fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed (Huse et al.(1989) Science 246: 1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

An antibody made according to the present invention can be used todetect the ORF1 (or ORF2) protein in or on cells, cell extracts, or inother biological preparations which can contain the ORF1 or ORF2protein. Additionally, such an antibody can be labeled with a detectormolecule to allow for detection of an antigen/antibody complex. Suitablelabels include various enzymes, fluorescent molecules, radioactivelabels, chemiluminescent molecules and the like. For example, enzymesuseful for labeling antibodies include horseradish peroxidase andalkaline phosphatase. Fluorescent labels include, but are not limitedto, fluorescein, rhodamine, dansyl chloride or phycoerythrin.

The viability of the two cell populations is determined, as described inExample 2 below, by mixing aliquots of the isolated cell populationswith propidium iodide that is taken up into the cell nuclei only bynon-viable cells. The stained cells are analyzed by flow cytometry toobtain total cell counts, as shown for example, in FIG. 2, as well asthe proportions of live and dead cells in the sample population, asshown in FIG. 3.

In addition to the host cell viability measurements, the methods of theinvention further comprise the steps of contacting the isolated cellpopulations with antibodies specific for the viral antigens ORF1 andORF2, also as described in Example 2 below. The cells are subsequentlywashed and may be contacted with anti-IgG antiserum labeled with afluorophore such as, but not limited to, FITC before measuring the cellbound florescence by a flow cytometer, also as described in Example 2.

Exemplary viability results for the growth of PCV2 virus on PK-15 cellsgrown in a liter batch culture with fetal calf serum in the medium areshown in FIGS. 2, 3 and 4. For example, in one experiment after fivedays incubation, about 50% of the total cultured cell population werelocated in the medium supernatant and were predominantly dead. Thiscontrasted with the remaining 50% of the cells which were adherent tothe CYTODEX™ (Amersham Biosciences, Inc) substratum and which wereviable.

Measurement of the viral production in the batch cultures containingfetal calf serum showed that ORF1 was transitorily expressed during days1 and 2, and only in the substratum adhered cells. ORF1 was detected inthe supernatant (or non-adherent) cells only from days 3 to 5 as shown,for example, in FIG. 6. The viral capsid antigen ORF2 was detectableonly in the supernatant, non-adherent, cells with a continuous increaseover the incubation period after the first day of incubation such asshown in FIG. 6.

The method of the invention allows the detection and monitoring of acircovirus and infected cells by detecting ORF1 and ORF2 expressed insupernatant (and therefore predominantly dead) cells until the harvestpoint. The rapidity with which these data are obtained permits frequentperiodic monitoring of the progress of the infection of the cellculture. The culture may then be harvested at a point that gives a highyield of virus suitable for seeding a large batch host cell culture forthe ultimate production of virus of sufficient quality and quantity foruse in, for example, vaccine development and production.

The seed cultures developed as a result of the use of the methods of theinvention can be used to seed large volume cell cultures, in the orderof 100-1000 liter volumes. In this procedure, the seed culture harvestedat day 5 or other time point as particularly indicated by ORF2expression as determined above, is inoculated into a large-scale cellculture comprising cell culture medium containing fetal calf serum. Theviability of the host cells, such as PK-15 cells, is then monitoredaccording to the methods described in Example 2 below. After an initialincubation period which may be, but is not limited to, about 3 days, thecell culture medium can be exchanged with medium that does not includefetal calf serum, and the incubation is continued, again with periodicin-process sampling and determination as shown, for example, in FIG. 7.

Another aspect, therefore, of the invention is the in-process monitoringof the production of circovirus in large scale host cell cultures bymonitoring the production of ORF1 and ORF2 using FACS. Using the methodsof the invention as described in Example 2, the cell cultures may bemonitored rapidly and more frequently when compared to conventionalculture-based procedures so that the culture may be harvested once adesired viral yield has been attained. The cultures can be monitored forthe expression of the viral-specific antigens ORF1 and ORF2 as shown,for example, in FIG. 7. Typically, the production of total ORF1 and ORF2antigen, as shown in FIG. 7, mimics the pattern of host cell growth asillustrated in FIG. 7. For example, as described in Example 5 below, themarkers ORF1 and ORF2 of PCV2 grown on PK-15 cells increase linearlyfrom day 4 to day 7 and display similar patterns of increase for bothadherent cells attached to a substratum and to the non-adherent cells inthe medium supernatant.

Removal of the serum from the culture medium reduces and delays theonset of the viral antigen expression, particularly noticeable if theculture is harvested at day 5. Continuation of the culture beyond day 5(as determined from the point of the seeding of the culture) can,however, significantly increase the yield of ORF2 antigen, that is, ofmature viral particles (see FIG. 7).

This invention, therefore, provides a FACS procedure for the in-processmonitoring and rapid determination of the useful harvesting point of acell culture infected with a circovirus such that an optimum yield ofthe virus can be obtained. The methods comprise providing a seed cultureof circovirus infected host cells and inoculating a batch culturetherewith, incubating the seed culture, removing aliquots of thecultured cells, separating supernatant cells and adherent cells,releasing the adherent cells from their substratum, determining theviability of the host cells, determining the percentage of ORF1 and ORF2in the cell sample by FACS, and determining the harvesting point of theculture.

One aspect of the invention provides a fluorescent antibody cell sorting(FACS)-based method for detecting the production of circovirus antigenby a culture of viral infected host cells, wherein the production ofcircovirus antigen is related to the percentage of open reading frame 1(ORF1) and open reading frame 2 (ORF2) positive cells, comprisingisolating (i) non-adherent host cells and (ii) adherent host cellsreleased from the substratum from a sample comprising a population ofnon-adherent host cells and a population of host cells adhering to asubstratum from a host cell culture infected with a circovirus, anddetermining (i) the percentage of open reading frame 1 (ORF1)-positivecells and (ii) the percentage of open reading frame 2 (ORF2)-positivecells present in the isolated non-adherent host cells and the adherenthost cells released from the substratum.

In various embodiments of this aspect of the method, the circovirus canbe PCV2. In various embodiments, the host cell strain can be PK-15. Itis contemplated, however, that the methods of the invention can beusefully applied for detecting and in-process monitoring of theproduction of any circovirus cultured on isolated mammalian host cellsand for which there are available viral antigen specific antibodies.

Another aspect of the invention provides a fluorescent antibody cellsorting (FACS)-based method for in-process monitoring of the productionof a circovirus from cultured host cells, wherein plotting changes inlevels of viability, ORF1 and ORF2, determines a time course ofcircovirus production in a host cell culture comprising isolating (i)non-adherent host cells and (ii) adherent host cells released from thesubstratum from a sample comprising a population of non-adherent hostcells and a population of host cells adhering to a substratum from ahost cell culture infected with a circovirus, determining viability ofthe non-adherent host cells and adherent host cells, and determining (i)the percentage of open reading frame 1 (ORF1)-positive cells and (ii)the percentage of open reading frame 2 (ORF2)-positive cells present inthe isolated non-adherent host cells and the adherent host cellsreleased from the substratum.

In one embodiment of this method of the invention, the viability of thecells is determined by measuring propidium iodide uptake using flowcytometry.

In the various embodiments of this method of the invention, thecircovirus can be PCV2 and the host cell strain can be PK-15, althoughthe methods should not be construed as to be applicable solely to thisstrain of circovirus/host cell combination. For example, the methods ofthe invention may be generally applicable to chicken anemia virus orbeak and feather disease virus and host cells such as a suitable avianhost cell and the like.

Yet another aspect of the invention is a method of producing circovirusin serum-free conditions and, therefore, useful for the preparation of avaccine, comprising the steps of preparing a seed culture of acircovirus by in-process monitoring of the expression of ORF1 andseeding a host cell culture with a seed culture of a circovirus,incubating the host cell culture in the absence of fetal calf serum,monitoring (i) viability of the host cells, and (ii) ORF1 and ORF2expression by the host cells, and harvesting the circovirus from thehost cell culture when ORF2 expression is approximately the same in thenon-adherent cells and adherent cells of the host cell culture.

In various embodiments of this aspect of the invention, the yield ofcircovirus in the seed culture may be determined by monitoring theexpression of circovirus ORF1 and ORF2 antigens in supernatant hostcells using a fluorescent antibody cell sorting (FACS)-based method, andwherein the seed culture is harvested when ORF2 is expressed by thenon-adherent cells.

In the various embodiments of this aspect of the invention, theviability of the cells may be determined by measuring propidium iodideuptake using flow cytometry.

In the various embodiments of this method of the invention, theexpression of ORF1 and ORF2 antigens in a viral infected host cellculture can be monitored using a fluorescent antibody cell sorting(FACS)-based method, wherein the production of circovirus antigen isrelated to the percentage of open reading frame 1 (ORF1) and openreading frame 2 (ORF2) positive cells FACS.

In the various embodiments of this method of the invention, thecircovirus can be PCV2 and the host cell strain can be PK-15, althoughthe methods should not be construed as to be applicable solely to thisstrain of circovirus/host cell combination. It is contemplated, however,that the methods of the invention can be usefully applied for detectingand in-process monitoring of the production of any circovirus culturedon isolated mammalian host cells and for which there are available viralantigen specific antibodies.

It should be understood that the present invention is not limited to thespecific compositions, equipment or methods described herein and thatany composition having a formula or method steps equivalent to thosedescribed falls within the scope of the present invention. The methodsteps for determining the percentage of infected cells in a cell cultureare merely exemplary so as to enable one of ordinary skill in the art tomake the composition and use it according to the described process andits equivalents. It will also be understood that although the form ofthe invention shown and described herein constitutes preferredembodiments of the invention, it is not intended to illustrate allpossible forms of the invention. The words used are words of descriptionrather than of limitation. Various changes and variations may be made tothe present invention without departing from the spirit and scope of theinvention.

The invention is further described by the following non-limitingexamples:

EXAMPLES Example 1 Correspondence between the Designations of ORFs ofCircovirus PCV2

The ORFs of circovirus PCV2, their equivalent designators, and therespective sources thereof, incorporated herein by reference in theentirety, are shown in Table 1 below. ORF1 and ORF2, as described byMeehan et al. (1997; 1998) have been alternatively designated as ORFs 4and 13 respectively.

TABLE 1 PCV2 ORF numbering and equivalents Example 2: Assay ofviability, Detecting ORF1 and ORF2 in PCV2-infected PK-15 cells ORFNumbering Alternative Designations Meehan et al. U.S. patent U.S. PatentSer. Nos. J. Gen. Virol. application Ser. 6,368,601, 6,391,314, 78:221-227 No. 20020106639 6,660272, 6,217,883, Source (1997) to Wang etal. 6,517,843, 6,497,883 1 1 4 2 6 13 3 2 7 4 3 10 5 4 5 6 5 3 7 1 8 2 96 10 8 11 9 12 11 13 12

(a) Determination of Cell Counts and Cell Viability

Propidium iodide was used to assess plasma membrane integrity. Propidiumiodide is a fluorescent vital dye that stains nucleic acid. Dead cellsincorporate propidium iodide which was detected as a red stain by flowcytometry using a Galaxy cytometer (other similarly functioning modelsof flow cytometer may be used). This cytometer has the capability todifferentiate, detect and count cells that are unstained (viable) orpropidium iodide stained (dead). The number of cells in a volume of 200μl is determined according to the manufacturer's instructions for theparticular model of cytometer used.

1 ml of cell suspension was prepared in PBS in a tube specific for theGalaxy cytometer. The dilution of the cell suspension was adjusted tohave between about 2×10⁴ to about 1×10⁶ cells per ml (corresponding tothe linearity value of the cytometer count). To the cell suspension wasadded 5 μl of propidium iodide (50 μg/ml). The suspension was thenvortex mixed for several seconds and analyzed immediately on thecytometer. Typical settings for the cytometer were: Threshold onFSC-lineage scale; Fluorescence of cells detected on FL3 log scale(corresponding to the propidium iodide channel). The cytometer softwaregave the results of the counts automatically as dot plots such as shownin FIGS. 2 and 3.

(b) Determination of Percentage of Infected Cells by ORF1 and/or ORF2Measurement

Approximately 3×10⁶ cells were required for a single assay, aliquoted as1×10⁶ for a negative control, 1×10⁶ for the ORF1 detection and 1×10⁶ forthe ORF2 detection. For a different quantity of cells, the reagentvolumes were adapted accordingly.

(i) Fixation: BD CytofixCytoperm (BD Biosciences, ref. 554714) was used.Fixation was performed according to the recommendations of the fixativemanufacturer. Briefly, 3×10⁶ cells were centrifuged for 6 mins at 400 gin a 15 ml or 50 ml conical tube. The supernatant was discarded, thecells were resuspended with 750 μl of Cytofix and incubated for 20 mins.on ice. The cells were washed twice with 1 ml of PBS containing 1% BSAand resuspended in 1 ml of PBS/i % BSA. Samples were stored at 5°Celsius for up to 15 days before staining(ii) Staining for ORF1 and ORF2: The fixed cells were centrifuged for 6mins at 400 g, the supernatant discarded and 300 μl of 1×BD Penn/Washsolution was added. 100 μl of fixed cells was dispensed into 3 wells of96 microwell plates and centrifuged for 6 mins. at 400 g. Thesupernatant was discarded and 100 μl of 1×BD Perm/Wash solution wasadded. 5 μl of an antibody (1 mg/ml) was added to each well. Typically,5 μg of monoclonal antibody-purified Mab anti-PCV2 (ORF1) No 1991D3GA,Initial Concentration=1 mg/ml or purified Mab anti-PCV2 (ORF2) No1903A8BC, Initial Concentration=1 mg/ml was sufficient for each stain.Purified Mab anti-clostridium N°101B9B or equivalent, InitialConcentration=1 mg/ml, was used as a negative control.

The wells were incubated for 30 mins. on ice. The cells were washedtwice with 1×BD Perm Wash solution (200 μl/wash/well). Then was added100 μl of 1×BD Perm Wash solution containing 1 μg of anti mouse FITC(anti-mouse IgG conjugated with FITC (for example, Beckman, ref no.115-095-146) or the equivalent thereof) per well and the wells incubatedagain for 30 mins. on ice. The cells were washed twice with 1×BD PermWash solution (200 μl/wash/well) and resuspended in 200 μl of PBS/1% BSAper well.

(iii) Detection by FCM: Settings parameters for the cytometer, such as aGalaxy Cytometer (Partec), were typically: Threshold on FSC-lineagescale; Fluorescence of cells detected on FL1 log scale (corresponding tothe FITC channel).

Representative histograms showing in the fluorescence activity due todetection of ORF1 in a population of infected PK-15 host cells, comparedto a negative control population of cells, is shown in FIG. 3. Tocalculate the percentage of infected cells, the percentage of negativecells was subtracted from the percentage of ORF cells. In the left-handhistogram, there were 0% infected cells, whereas in the right-handexample of FIG. 3, 88% of the cells were infected.

A typical distribution of live and dead cells as determined bygranulometry is shown in FIG. 2 and a shift in the signal of propidiumiodide fluorescence due to the death of an infected host cell populationis shown in FIG. 4. In this case, 80% of the host cells were dead.

Example 3 PK-15 Count and Viability after PCV2 Inoculation in Batch SeedCultures

As shown in FIG. 3, At day 5 of the incubation period of a batch seedculture of PCV2 virus growing on PK-15 cells, 50% of the host cells werelocated in the supernatant and were mostly dead (73%). In contrast, 50%of the host cells were located on the CYTODEX™ (Amersham Biosciences,Inc) substratum support and were almost entirely a viable population(96%).

PCV2-infected PK-15 cells were detected at the end of the culture at day5, but only in the supernatant cells using flow cytometry to detect ORF1and ORF2. As shown, for example, in FIG. 6, the kinetics of theviral-specific antigen formation was dependent on the ORF: ORF1 wasproduced early and at low levels whilst ORF was produced later in theincubation cycle and at increasing levels.

Example 4 ORF1 and ORF2 Total Staining

As shown in FIG. 6, for ORF1 there was no signal on cells membranedetected during the later phase of the culture incubation period. Table2, below, shows that no membrane signals were detected on cells adherentto CYTODEX™ (Amersham Biosciences, Inc). Major ORF-specific signals wereon cells from the culture supernatant and were increasing from day 2 today 5. Results were similar for live or dead cells, with a maximum of60% at day 4.

TABLE 2 Count (total/ml) Cells from supernatant Adherent cells % of 3.6× 10⁵ Membrane + 4.7 × 10⁵ Membrane + Total Cells 43% Intracellular 57%Intracellular Staining Membrane compartments. Membrane compartments.ORF1 <3% 35% <3% <3% ORF2   37%  60% <LOQ <LOQ

Example 5 Culturing of PCV2 Circovirus on PK-15 Cells in Large ScaleBatch Culture with Serum Removal after 3 Days

A 300-liter fermenter was seeded with a culture of PK-15 cellspreviously incubated for 5 days with PCV2 virus, in accordance withdocuments cited herein. After 3 days of culturing, the medium wasexchanged for a growth medium lacking fetal calf serum. Samples taken ateach day were tested for PK-15 viability and the expression of ORF1 andORF2 as described in previous examples.

The time courses of the expression of the viral antigens ORF1 and ORF2is illustrated in FIG. 7.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theappended claims is not to be limited by particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope thereof.

1-9. (canceled)
 10. A method for monitoring circovirus production in ahost cell culture infected with circovirus, the method comprising: a)monitoring expression of ORF1 and/or ORF 2 in adherent and non-adherenthost cells using a fluorescent antibody cell sorting (FACS)-basedmethod; and b) plotting expression of ORF1 and/or ORF 2 over time;thereby monitoring circovirus production in the host cell culture. 11.The method according to claim 10, further comprising monitoringviability of host cells infected with circovirus using flow cytometry.12. The method according to claim 10, wherein the circovirus is PCV-2.13. The method according to claim 10, wherein the host cell is PK-15.